Manganese superoxide dismutase cloning and expression in microorganisms

ABSTRACT

Methods and compositions are provided for the production of human manganese superoxide dismutase and a protocol for enhancing efficiency of expression. The gene encoding for human manganese superoxide dismutase was isolated and inserted into a vector in conjunction with a synthetic linker which provides for enhanced efficiency in translation.  
       E. coli  strain HB101 containing the plasmid Nco5AHSODm was deposited at the A.T.C.C. on Oct. 3, 1986 and given Accession No. 67191.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

[0001] The production of the enzyme manganese human superoxide dismutase(hSODm) by recombinant DNA techniques is described. Both natural andmodified enzymes are produced utilizing novel DNA constructs, plasmidsand transformed microbial expression systems.

[0002] Superoxide dismutase (“SOD”) is in fact a variety of differentenzymes found in most living organisms. One function in mammals is todestroy superoxide. Superoxide is a material naturally produced duringphagocytosis and aerobic metabolism. The superoxide dismutases arecharacterized in families based on the metal ion associated with theenzyme, where the ions can be iron, manganese, copper, and copper andzinc. Superoxide dismutase, e.g., from bovine liver, has found clinicaluse, particularly as an anti-inflammatory agent in mammals includinghumans and to decrease tissue injury due to reperfusion (post-ischemic).Other utilities include scavenging superoxide anions due to exposure ofa host to various superoxide-inducing agents, e.g. radiation, paraquat,etc.; prophylaxis or therapy for certain degenerative diseases, e.g.,emphysema; food preservation; and the like.

[0003] It is therefore important that stable supplies of physiologicallyacceptable superoxide dismutase be made available, particularly for usein vivo as an anti-inflammatory agent or for other therapeutic purposes.For human application it would be preferable to employ the homologousenzyme to prevent or minimize possible immune response. By employingrecombinant DNA techniques, there is the opportunity to produce productsefficiently, which have the desired biological activities of superoxidedismutase, such as immunological and enzymatic activities.

Description of Relevant Publications

[0004] The primary structure of human liver manganese superoxidedismutase was described by Barra et al., J.B.C., 259:12595-12601,(1984). Either bacterial iron superoxide dismutase (FeSOD) or bacterialmanganese superoxide dismutase (MnSOD) were shown to be required as adefense against oxygen toxicity by Carlioz et al., EMBO Journal,5:623-630, (1986). The amino-terminal processing of methionine by yeastwas shown by Tsunasawa et al., J.B.C., 260:5382-5391, (1985). Humancopper/zinc superoxide dismutase was described by Hallewell, et al.,Nucleic Acids Research, 13:2017-2034, (1985). A superoxide dismutaseproduced in Serratia marcescens is described in EPO application 0172577.An immobilized superoxide dismutase was described in EPO application070656.

[0005] The amino acid sequence of human erythrocyte Cu—Zn superoxidedismutase was described in Jabusch et al., Biochemistry (1980)19:2310-2316 and Barra et al., FEBS Letters (1980) 120:53-55. Bovineerythrocyte Cu—Zn SOD was described by Steinman et al., J. Biol. Chem.(1974) 249:7326-7338. A SOD-1 (Cu—Zn SOD) cDNA clone is described byLieman-Hurwitz et al., Proc. Natl. Acad. Sci. USA (1982) 79:2808-2811.

SUMMARY OF THE INVENTION

[0006] Novel compositions are provided comprising nucleic acid sequencesfor the expression of polypeptides exhibiting the biological propertiesof human manganese superoxide dismutase (“ThSODm”). Also provided aremethods for producing such polypeptides employing recombinant DNAtechniques and microorganism hosts. The subject polypeptides find use invivo and in vitro in destroying superoxide.

[0007] Also provided is a novel modified hSODm. A modified DNA codingfor hSODm with the first two amino acids, lysine and histidine, removed,resulted in a polypeptide with the third amino acid, serine, positionedadjacent to the translation initiating amino acid methionine. Thismodified hSODm permitted removal of the amino terminal methionine byprocessing enzymes.

[0008] A method of treating a patient having inflammatory joint diseaseor post-ischemic tissue injury is also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 is a flow chart describing the construction of pNco5AhSODm,a bacterial expression plasmid for hSODm.

[0010]FIG. 2 shows the junction sequence between vector and insert andtranslation initiation sites for the mini-gene and hSODm in pNco5AhSODm.

[0011]FIG. 3 is a flowchart describing the construction ofpC1/1XSShSODm, a yeast expression plasmid for hSODm.

[0012]FIG. 4 indicates the sequence of both the coding strand of hSODmcDNA (5′-3′) and the amino acid sequence of the hSODm translationproduct with signal sequence.

[0013]FIG. 5 is a picture of an SOD activity gel depicting expression ofhSODm in bacteria.

DESCRIPTION OF SPECIFIC EMBODIMENTS

[0014] Methods and compositions are provided for the efficientexpression of polypeptides demonstrating the biological activities ofhuman manganese superoxide dismutase (“hSODm”). The methods employ a DNAsequence (“hSODm gene”) encoding at least a substantial portion or allof the amino acid sequence of hSODm in conjunction with atranscriptional initiation regulatory region providing for efficientexpression in the expression host. The hSODm gene is inserted into anappropriate vector for expression in a host, conveniently underconditions which allow for processing to remove the N-terminalmethionine.

[0015] It is possible to modify the 5′-end of the structural gene toremove one or both of the first two amino acids of the maturepolypeptide. Such modification may be accomplished by a variety ofconventional methods. For example, the structural gene may be restrictednear its 5′-end to remove a known number of nucleotides. A syntheticoligonucleotide (adapter) may then be joined to the cohesive endremaining after restriction. The oligonucleotide will restore andsubstitute the base pairs as necessary to provide the desired amino acidsequence. Alternatively, site-specific mutagenesis employing, e.g.,phage M13, or primer repair can be used to effect an appropriatemodification to the 5′-end of the structural gene.

[0016] In order to prepare hSODm, it is necessary to have a DNA sequencewhich encodes for hSODm. One manner of achieving such sequence is toclone cDNA from messenger RNA from cells which produce hSODm.Conveniently, human kidney cells may be used for this purpose. After thecDNA is cloned, where the DNA coding sequence is unknown, but at least apartial amino acid sequence is known, one may then screen the cDNA withmixtures of complementary synthetic DNA probes having all, orsubstantially all, of the possible variations of nucleotides encodingfor the particular series of amino acid residues. The choice of theoligopeptide fragment for which the sequence encodes is somewhatarbitrary, although the fragment chosen will usually be selected tominimize the number of different sequences which must be synthesized.Two or more such probes hybridizing with the two ends of the hSODm EDNAmay be used to select for those sequences containing all orsubstantially all of the coding sequence.

[0017] For hSODm, conveniently a DNA sequence encoding for the aminoacid residues 197 to 211 can be used, particularly a probe having atleast about 15 bases and not more than about 60 bases, more convenientlyabout 44 bases. With the probe in place one may then use restrictionenzymes to digest the clones which appear to hybridize with the labeledprobes, fractionate the DNA fragments and repeat the hybridization,particularly by employing a second series of probes which hybridize toDNA sequences encoding for a different series of amino acid residues inhSODm. Conveniently, these amino acid residues may be 23 to 27. Suchprobes are shown in Example I. One or more clones may be found which arepositive to both probes and these may be used as a source for cDNAencoding for at least a substantial proportion of hSODm.

[0018] Quite surprisingly, it was found that the amino acid sequencewhich had been published for hSODm differed from the amino acid sequenceencoded for by the cDNA. Specifically, two amino acids in positions 124and 125 differed. Barra et al. disclosed the sequence of the matureprotein including a leucine 124 and a glycine 125, while, the cDNA codedfor glycine 124 and a tryptophan 125.

[0019] An mRNA source of human manganese superoxide dismutase cDNA canbe isolated from any human cell producing manganese superoxidedismutase, for example kidney cells. However, one convenient source ofsuch cDNA is the human kidney cell libraries wherein the entire mRNA hasbeen converted to cDNA and inserted into lambda bacterial phage (λgt10).(Huynh et al., 1985) Individual specific clones can be selected using aprimer which selects the particular clone containing the hSODm cDNA.Selection is by hybridizing with oligonucleotide probes that have beensynthesized based on known amino acid sequences. The sequences of theprobes were produced based upon the amino acid sequence of Barra et al.,1984, (supra). The sequence of DNA oligonucleotides illustrated in FIG.4 codes for the entire amino acid sequence of the human manganesesuperoxide dismutase plus the 25 amino acid signal sequence which isnormally removed during transfer of the nascent protein from thecytoplasm to the mitochondria. Based upon this DNA sequence, the correctamino acid sequence for the hSODm was determined. As previouslydiscussed there were two amino acids, glycine 124 and tryptophan 125that were previously not known.

[0020] Bacterial expression vectors may be constructed by insertingeither the entire hSODm EDNA or the modified hSODm cDNA coding for hSODmwith the first two amino acids removed. Any bacterial expression systemmay be used with these DNA constructs. A preferred plasmid expressionsystem is the plasmid pNco5A. In one embodiment the hSODm cDNA wasinserted after a mini-gene which coded for 4 amino acids. This mini-genewas located such that it occurred in a Shine-Dalgarno region andprovided the proper sequence for the Shine-Dalgarno region. Thetranslation of the mini-gene thereby facilitated improved translation ofthe hSODm gene.

[0021] The preferred expression plasmid pNco5AhSODm was constructedusing a synthetic linker described in FIG. 1. This linker incorporatesin the same sequence both the Shine-Dalgarno region and a mini-geneterminating in a stop codon joined to the met codon of the modifiedhSODm. For the construction of the modified hSODm expression system, thecodons for the two initial amino acids (Lys and His) were deleted. As aresult, the initiation codon (Met) was located adjacent to the thirdamino acid (Ser) of the hSODm. The processing enzymes found in bacteriaand yeast recognize a methionine adjacent to a serine and remove it asdescribed by Tsunasawa et al., JBC 260:5382-5391 (1985) yielding amature protein free from N-terminal methionine. This processing does notoccur if the methionine is next to lysine or histidine (the first andsecond residues of HSODm). The serine adjacent to the initiatingmethionine in the hSODm polypeptide may also be replaced by any one ofthe following amino acids: alanine, glycine, proline, threonine, andvaline. Any of these amino acids adjacent to the initiating methioninewill result in a hSODm polypeptide which will be processed by thecellular amino-terminal processing enzymes resulting in removal of themethionine. The removal of the N-terminal methionine reduces thelikelihood of an immunogenic reaction when the recombinant DNA productis administered to a human subject.

[0022] If desired, the hSODm gene may be joined to signal sequences,secretory leader and processing signals, to provide for secretion andprocessing of the hSODm. Various secretory leader and processing signalshave been described in the literature. See for example, U.S. Pat. Nos.4,336,336 and 4,338,397, as well as copending application Ser. Nos.522,909, filed Aug. 12, 1983 and 488,857, filed Apr. 26, 1983, therelevant portions of which are incorporated herein by reference.

[0023] Of particular interest as hosts are unicellular microorganismhosts, both prokaryotes and eukaryotes, such as bacteria, algae, fungi,e.g. yeast, etc. In particular, E. coli, B. subtilis, S. cerevisiae, K.lactis, Streptomyces, and Neurospora may afford hosts.

[0024] A wide variety of vectors are available for use in unicellularmicroorganisms, the vectors being derived from plasmids and viruses. Thevectors may be single copy or low or high multicopy vectors. Vectors mayserve for cloning and/or expression. In view of the ample literatureconcerning vectors, commercial availability of many vectors, and evenmanuals describing vectors and their restriction maps andcharacteristics, no extensive discussion is required here. As iswell-known, the vectors normally involve markers allowing for selection,which markers may provide for cytotoxic agent resistance, prototrophy orimmunity. Frequently, a plurality of markers are present, which providefor different characteristics.

[0025] In addition to the markers, vectors will usually have areplication system and in the case of expression vectors, will usuallyinclude both the initiation and termination transcriptional regulatorysignals, such as promoters which may be single or multiple tandempromoters, an mRNA capping sequence, a TATA box, enchancers, terminator,polyadenylation sequence, and one or more stop codons associated withthe terminator. For translation, there will usually be a ribosomalbinding site for prokaryotes, as well as one or more stop codons, whichmay be provided by the vector or by the structural gene. Alternatively,these regulatory sequences may be present on a fragment containing thestructural gene, which is inserted into the vector.

[0026] Usually, there will be one or more restriction sites convenientlylocated for insertion of the structural gene into the expression vector.Once inserted, the expression vector containing the structural gene maybe introduced into an appropriate host and the host cloned providing forefficient expression of hSODm.

[0027] In some instances, specialized properties may be provided for thevector, such as temperature sensitivity of expression, operators oractivators for regulation of transcription, and the like. Of particularinterest is the ability to control transcription by exogenous means,such as temperature, inducers, corepressors, etc., where transcriptioncan be induced or repressed by an exogenous compound, usually organic.

[0028] In some instances integration of the expression system may bedesirable. In that event that expression construct will usually involvea sequence homologous with DNA in a chromosome of the host. Frequently,the DNA may be a gene which provides prototrophy to an auxotrophic hostor provides some other means for selection.

[0029] A number of expression systems have been developed for yeastwhich provide for constitutive or regulated expression. See, forexample, EPA 20 83/306,507.1 and U.S. application Ser. No. 868,639filed, May 29, 1986, whose disclosure is incorporated herein byreference.

[0030] Strong yeast transcriptional initiation regions includeglycolytic enzyme initiation regions of genes such asglyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, alcoholdehydrogenase, phosphoglucoseisomerase, triose phosphate isomerase,phophofructokinase, etc., acid phosphatase, etc. Regulatory and/orenhancer regions may be joined 5′ to the transcription initiationregion, which regions may be obtained from UDP-galactose epimerase(GAL10), galactokinase (GAL1), acid phosphatase (PH05), alcoholdehydrogenase I and II, etc.

[0031] Where the hSODm is made intracellularly, when the cell culturehas reached a high density, the cells may be isolated, conveniently bycentrifugation, lysed and the hSODm isolated by various techniques, suchas extraction, affinity chromatography, electrophoresis, dialysis, orcombinations thereof. Where the product is secreted, similar techniquesmay be employed with the nutrient medium, but the desired product willbe a substantially higher proportion of total protein in the nutrientmedium than in the cell lysate.

[0032] The hSODm which is formed has substantially the same amino acidsequence as the naturally occurring human managanese superoxidedismutase, usually differing by no more than 5 amino acids, more usuallydiffering by no more than 3 amino acids, and differing in one example byonly 2 amino acids. The recombinant hSODm (“r-hSODm”) displayssubstantially the same biological properties as naturally occuringhSODm. The biological properties include immunological properties, whereantibodies raised to authentic hSODm cross-react with r-hSODm.Furthermore, in common bioassays employed for hSODm, the r-hSODm productdemonstrates a substantial proportion, usually at least about 10%,preferrably at least about 50%, more preferably at least 80%, of theenzymatic activity of the authentic hSODm, based on weight of protein.An illustrative assay technique is described by Marklund et al., Eur. J.Biochem. (1974) 47:469-474.

[0033] Human manganese superoxide dismutase was produced in bacterialcells using the plasmid containing the isolated hSODm gene. The activityof the superoxide dismutase in the bacterial cells was determined bypolyacrylamide gel electrophoresis and staining for superoxide dismutaseactivity as described by Beauchamp et al. (1971). In Example III thereis a comparison of the human superoxide dismutase produced in thebacterial transformant with human manganese superoxide dismutase,bacterial iron superoxide dismutase and a hybrid between the bacterialmanganese and iron superoxide dismutase.

[0034] Yeast expression vectors for the production of the humanmanganese superoxide dismutase may be constructed beginning with theplasmid pC1/1 as described in the European patent application 116 201.Synthetic DNA linkers were constructed which had restriction sites withcomplementary ends to a restriction site in the plasmid pC1/1. Theselinkers were inserted into the plasmid using conventional ligationprocedures. One such linker is shown in Example IV. These linkers areuseful in providing unique restriction sites for constructing expressionplasmids such as pC1/1XSS. The GAP regulatory regions were isolated fromthe plasmid pPGAP as described in European patent application 164556.The GAP promoter and the GAP terminator were isolated and combined usingthe restriction sites present in the linker inserted into pC1/1 XSS. Thehuman manganese superoxide dismutase gene was inserted into the yeastexpression vector pC1/1 XSS GAP using a linker described in Example IV.The resulting plasmid was pC1/1 XSS GAP hSODm which was used totransform the S. cerevisiae strain P017 which then expressed the humansuperoxide dismutase. Human superoxide dismutase activity produced inyeast is determined by the same procedures used for the bacterialexpression system.

[0035] Human superoxide dismutase is useful in applications wheresuperoxide dismutase is useful. It is particularly useful in humanapplications to prevent or minimize possible immune response. Humansuperoxide dismutase finds clinical use particularly as ananti-inflammatory agent and to minimize post-ischemic tissue injury.

[0036] Wilsmann, Procedings of the 4th International Conference onSuperoxide and Superoxide Dismutase held in Rome, Italy, Sep. 1-6, 1985,ed. Rotilio pp 500-507 describes 10 years of clinical experience withSOD treatment of inflammatory disorders, particularly inflammatory jointdisease. Flohe et al., pp 424-430 in Biological and Clinical Aspects ofSuperoxide and Superoxide Dismutase Developments in Biochemistry, v.11(b), Bannister and Bannister, (eds.), 1980, describes clinical trialsusing superoxide dismutase to treat osteoarthritis of the knee joint.Both articles recognize that human SOD would be preferable to bovine SODto prevent allergic/anaphylactic reactions in the patients.

[0037] Patients having inflammatory joint disease are treated by aweekly intraarticular injection into a joint afflicted with the diseaseof a solution having human manganese superoxide dismutase in a suitablediluent in an amount effective to reduce inflammation, usually 1 to 10mg, more usually 2 to 6 mg. The injections are given weekly for a periodof time sufficient to reduce inflammation, usually for 2 to 8 weeks,more usually for 4 to 6 weeks. Because the articular capsule limitsleakage of the high molecular weight compound each afflicted jointshould be treated with the required dosage.

[0038] hSOD is conveniently stored lyophilized with sugar, usuallysucrose, usually in a ratio of 1:2 w/w. The lyophilized enzyme isconveniently reconstituted in a suitable diluent for the particularapplication. For example, to treat inflammatory joint disease hSOD maybe reconstituted in physiologic saline in a volume convenient forintraarticular administration so that the required number of milligramsof hSOD are contained within 0.5 to 5 ml of solution.

[0039] hSOD is also useful to minimize post-ischemic tissue damage. Suchdamage occurs whenever perfusion of an organ is interrupted as by thepresence of a clot, due to a heart attack, or where blood flow to anorgan is interrupted during surgical procedures such as where bloodsupply to an organ is clamped off during organ transplant or othersurgery. In such instances the patient is administered 10 mg to 1,000mg, more usually 50 mg to 500 mg of human manganese superoxide dismutasein a suitable diluent during the isahemic reaction. When the patientsuffers ischemia due to a disease the solution is administeredintraveneously or intraarterially as a bolus dosage or a continuousinfusion. In such situations the hSOD may be administered in conjunctionwith fibrinolytic agents such as fibrin, fibrinogen or tissueplasminogen activator (TPA).

[0040] When ischemic damage is due to a surgical procedure, hSOD isadministered during surgery. This application finds particular use inorgan transplant surgery where hSOD is preferably administered prior toreirrigation of the organ and is also useful in any other surgery wherebloodflow to an organ is interrupted, such as open heart surgery.

EXPERIMENTAL

[0041] General Methods

[0042] Preparation of plasmid DNA and poly(A)+ RNA, restriction enzymedigestions, screening of cDNA and genomic libraries, agarose gelelectrophoresis, and DNA blotting and hybridization were carried out bystandard procedures as described by Maniatis et al, (1982) MolecularCloning: Lab. Manual (CSH New York: CSH Labs). DNA sequencing was doneby the dideoxy-chain termination method (Sanger et al., J. Mol. Biol.143:161-178) with specific oligonucleotide primers (Sanchez-Pescador etal., DNA (1984) 3:339-343) after suboloning into bacteriophage M13vectors (Messing, Methods in Enzymology (1983) 101:20-78). Syntheticoligonucleotides were synthesized as described in Warner et al, DNA(1984) 3:401-411).

[0043] Bacterial cells were transformed and grown in L Broth-ampicillinas described by Maniatis et al., (1982), supra. Yeast were transformedaccording to Hinnen et al., Proc. Natl. Acad. Sci. USA (1978) 75:1929-1933 and were grown using a variety of media including selectivemedium (yeast nitrogen base supplemented with amino acids, etc.) asappropriate but without leucine; yeast extract and peptone containing 2%(w/v) glucose (YEPD); and in the case of plating medium containing 2%(w/v) agar and for transformation 3% top agar.

Example I Cloning of hSODm, cDNA and Amino Acid Sequence Determination

[0044] cDNA Cloning

[0045] The cDNA clones encoding the human manganese SOD were isolatedfrom an adult human kidney library in λgt10 (Huynh et al. 1985 “in DNACloning: A Practical Approach”, Glover, D. H. ed. IRL Press Oxford, pp49-78). Double-stranded cDNA was prepared essentially as described byGubler et al., Gene (1983) 25:263-269 and after methylation of internalEcoRI sites and the addition of EcoRI linkers the cDNA was ligated intothe EcoRI site of λgt10. Clones containing segments of the humanmanganese SOD were identified by hybridization with two chemicallysynthesized oligonucleotide probes. The sequence of the probes is shownbelow and was deduced from the amino acid sequence by Barra et al.,J.B.C. (1984) 259:12595.

[0046] Probe 1:

5′-TTGTTCACGTAGGCGGCGTGGTGCTTGGAGTGGTGCAGCTGCAT-3′

[0047] (complementary to mRNA that codes for amino acids 23-27 of themature protein as numbered by Barra et al.)

[0048] Probe 2:

5′-GCCATGTATCTCTCGGTCACGTTCTCCCAGTTGATCACGTTCCA-3′

[0049] (complementary to mRNA that codes for amino acids 197-211 of themature protein as numbered by Barra et al.)

[0050] Probe 1 and Probe 2 were used to select those members of theλgt10 library that contained the hSODm gene. Probe 1 was specific forDNA coding for the hSODm amino acids 23 to 27, and Probe 2 was specificfor DNA coding for the hSODm amino acids 197-211. Those library clonesthat hybridized with both Probe 1 and Probe 2 contained either a largefragment (equivalent to amino acids 23-211) or the complete hSODm gene.One of the isolates was a full-length clone (hMnSOD-4). This clone(hMnSOD-4) was selected for sequencing and expression.

[0051] Sequence of hSOD Mn

[0052]FIG. 4 shows the full nucleotide sequence determined for clonehMnSOD-4 and the deduced amino acid sequence. In parenthesis areindicated the EcoRI linkers used in the cloning procedure. When thisamino acid sequence is compared to that reported by Barra et al. 1984supra, two differences emerge. First, Barra et al. presented thesequence of the mature protein which has a lysine at the amino terminus.The sequence shown in FIG. 4 codes also for an additional 25 amino acidsupstream from the lysine (which is in position 1, see FIG. 4). Theseamino acids correspond to a signal sequence that is cleaved to give riseto a mature protein. Second, there are two amino acids (Gly, Trp inpositions 124, 125; underlined in FIG. 4) that are absent from thepublished amino acid sequence by Barra et al. 1984 supra. Therefore, theobserved amino acid sequence is different from that previously reported.

Example II Construction of a Bacterial Expression Vector for Serine hSODMn: Plasmid pNco5AHSODm

[0053] (FIGS. 1a & b)

[0054] A bacterial expression vector was constructed which containshSODm cDNA inserted after a mini-gene (coding for 4 amino acids). Thetwo sequences are separated by a translational stop codon and theygenerate a polycistronic mRNA.

[0055] Plasmid pNco5AHSODm was constructed as follows. An EcoRI fragmentof about 850 bp from lambda clone, hMnSOD-4, coding for the pre-SODm wasexcised by EcoRI digestion and purified by agarose gel electrophoresis.This fragment was ligated to ptac5 (Hallewell et al., Nucleic Acids Res.(1985) 13:2017) previously digested with EcoRI. The EcoRI site ispresent in the polylinker of ptac5. The ligation mix was used totransform E. coli. Several transformant colonies selected inampicillin-L broth plates were screened by restriction analysis. Oneclone (ptac5HSODm) containing the correct orientation (3′-end near theSalI site on the polylinker) was selected for further manipulation.

[0056] Plasmid ptac5HSODm was digested with NarI which cuts in the hSODmcDNA sequences coding for amino acids 12-13. The NarI site is indicatedwith the double underlining in FIG. 4.

[0057] Synthetic linkers of the sequence described in FIG. 1 wereligated to linearized ptac5HSODm.

[0058] The linker provides for a NcoI overhang, a mini-gene coding for 4amino acids which incorporates in its sequence a Shine-Dalgarno sequencefor ribosome binding, a stop signal, an ATG for the first translationinitiation methionine codon for hSODm and a sequence coding for tenamino acids of the hSODm.

[0059] The two first amino acids of the mature protein were not includedin the synthetic linker (see FIGS. 1 and 4). Instead, the residue thatis in third position in the mature protein (Ser) is adjacent to the Metin the linker sequence. This choice is made because bacteria and yeastprocess and cleave after methionine when followed by a serine (Tsunasawaet al., J.B.C. (1985) 260:5382). Therefore, a mature hSODm is obtainedwithout an N-terminal methionine. If the initiating methionine isfollowed by Lys the first residue of the mature protein, the methioninewould not be cleaved. In this case one would obtain a methionyl-hSODmwhich could be antigenic for human use because it contained anN-terminal methionine.

[0060] The ptac5HSODm with ligated linkers was digested with NcoI andSalI (which cuts after the 3′-end of the insert). The NcoI-SalI fragment(ca.715bp) was gel purified. This fragment was cloned into NcoI-SalIdigested pSODNco5A to yield pNco5AHSODm.

[0061] Plasmid pNco5ASOD is a pBR322 derived bacterial expressionplasmid for Cu/Zn hSOD. The plasmid contains the tac promoter and Cu/ZnhSOD cDNA as an EcoRI-SalI insert substituting pBR322 sequences betweenEcoRI and SalI. The tac promoter is proximal to the EcoRI site and thedirection of transcription is clockwise. The tac promoter and hSOD cDNAinsert of pSODNco5A was obtained from pNco5ASOD (Hallewell et al.,supra). FIG. 2 shows the junction nucleotide sequence between vector andinsert and translation initiation region of both mini-gene and hSODm.The figure indicates the source of the DNA sequences.

Example III Expression of hSODm in Bacteria

[0062] Bacterial cells E. coli MC1061 (Casadaban et al., J. Mol. Biol.(1980) 138:179-207) or E. coli HB101 were transformed with plasmidpNco5AHSODm. The hSODm transformant cells were selected in L-brothplates containing 100 ug/ml of ampicillin. Cultures prepared inL-broth-amp containing 0.2 mM MnCl₂ were grown at 37° C. tolate-log-phase with agitation.

[0063] Cells (1 ml) were harvested and lysed with glass beads asfollows: Fifty ul of 2 mM MnCl₂, 35 mM KPO₄ pH 7.8, 1 mM PMSF were addedto the cell pellet. In addition, 50 ul of acid washed glass beads werealso added. The mixture was vortexed six times for 30 seconds each,leaving the tube 30 seconds on ice between vortexing. The mix wascentrifuged for 30 seconds in an Eppendorf microfuge. The supernatantwas saved and the pellet was discarded.

[0064] A sample of 10 ul of the supernatant was loaded onto a 10% nativepolyacrylamide gel electrophoresed and stained for SOD activity(Beauchamp et al., Anal. Biochem. (1971) 44:276-287.)

[0065] Results show that E. coli MC1061 or HB101 constitutivelyexpressed hSODm. Several bands are detected on the activity gels ofextracts of the bacterial tranformants. The bands correspond to thehuman manganese SOD, bacterial manganese SOD, bacterial iron SOD and ahybrid between bacterial manganese and iron SOD. The bacterial SOD formshave been previously described (Caroioz et al., EMBO. J. (1986) 5:623).

[0066] In order to confirm the identity of the newly synthesized hSODm,plasmid pNco5AHSODm was transformed into E. coli cells QC774(SOD A⁻ SODB⁻) (Caroioz et al., supra), which lack bacterial SOD. Extracts of thesecells were prepared as above and electrophoresed on a 10% nativeacrylamide gel as previously described. FIG. 5 shows the activity gel.The first lane contains 0.4 μg of purified Cu, Zn human hSOD as activitycontrol. Lane 2 contains extract of E. coli strain GL4468 (parent of theSOD⁻QC774 strain). Two bacterial SOD activity bands are detectedcorresponding to the following from top to bottom: E. coli SOD_(m) andE. coli FeSOD. Lane 3 contains extract of E. coli strain QC774transformed with pBR322 (negative control) and lane 4 contains extractof the same QC774 strain transformed with the SODm expression plasmid.The results clearly confirm that a new SOD activity band appears in theSOD⁻ mutants when transformed with the expression plasmid (lane 4). Thisband is not present in extracts of cells without plasmid (lane 3). Thenew band has a molecular weight of about 23,000 daltons as determined byCoomassie Blue staining of gels.

Example IV Construction of Yeast Expression Vectors for hSODm

[0067] Construction of Plasmid pC1/1 XSS

[0068] Since some SODm contain a BamHI site in their sequence, it wasnecessary to engineer the yeast expression vectors (in which the cloningsite for the expression cassette is a BamHI) to include other singlesites. For this reason, two derivatives were constructed in which thesingle vector BamHI site was deleted and new SacI, SacII and XhoI (XSS)were introduced. Description of the construction of these derivatives isshown below.

[0069] Plasmid pC1/1 (see EPO 116 201) was linearized with BamHI andphosphatased. The synthetic DNA linkers of sequence shown below, havingBamHI complementary ends and restriction endonuclease sites for SacI,SacII and XhoI were phosphorylated with T4 polynucleotide kinase. Afterremoval of the kinase they were ligated to the linearized pC1/1. TheBamHI is not reconstituted by ligation of this linker. 5′ GATCG AGCT|CCCGC|GGC|TCGAGC 3′      3′C|TCGA GGG|CG CCG AGCT|CGCTAG 5′         SacI    SacII   XhoI

[0070] After transformation of the yeast strain MC1061 a recombinantplasmid was obtained which had the linker inserted at the BamHI site.SacI, SacII and XhoI are unique sites in pC1/1 XSS.

[0071] Construction of Plasmid pPGAPXSS

[0072] Plasmid pPGAPXSS is a derivative of pPGAP (EPO 164 556) in whichthe following linker was inserted at the junction between the GAPpromoter and/or GAP terminator sequences and the vector sequences:           BglII   SacI   SacII   XhoI   BamHI BXSSBE-U 5′ GATCTGAGCT|CC GC|GGC|TCGA G|GATC CA 3′ BXSSBB-L 3′     AC|TCGA CG|CG CCGAGCT|C CTAG|GTCTAG 5′

[0073] As FIG. 3 shows, the linkers provide for SacI, SacII, XhoI andBamHI adjacent to both, 5′ end of GAP promoter sequences and 3′ end ofGAP terminator sequences. Both BamHI and BglII sites coming from pPGAPends and linkers, respectively were not reconstructed.

[0074] Construction of Plasmid pC1/1XSSGAP hSODm

[0075] for Expression of hSODm in Yeast (FIGS. 3a & b)

[0076] In order to prepare yeast expression vectors for hSODm, the polyAsequences present at the end of the hSODm cDNA were removed.

[0077] To remove the polyA sequences from pNco5AHSODm, the plasmid wasdigested with PvuI which cuts upstream from the polyA tract as shown inFIG. 4. The overhang was filled in using the Klenow fragment of DNApolymerase I. The plasmid was subsequently digested with NcoI. A 615 bpfragment containing the hSODm cDNA was gel isolated and was re-insertedinto pNco5AHSOD (see FIG. 1), digested with NcoI and SmaI (which cutsnext to SalI, as indicated in FIG. 1) to yield pNco5AHSODm-PA, aftertransformation of E. coli MC 1061.

[0078] Plasmid pNco5HSODm-PA (see FIG. 3) is linearised with NarI andligated to the synthetic DNA linker shown below. The lower strand of thelinker is phosphorylated in this ligation.    Met Ser Leu Pro Asp LeuPro Tyr Asp Tyr Gly 5′ ATG TCT TTG CCA GAC TTG CCA TAT GAC TAG GG 3′    3′ AGA AAC GGT CTG AAC GGT ATA CTG ATG CCG C 5′

[0079] Following ligation, the plasmid is cut with SalI and the about720 bp linker to SalI fragment is isolated by preparative agarose gelelectrophoresis. This fragment is ligated to NcoI and SalI cut PPGAPXSSin the presence of NcoI. After transformation of MC1061 a colonycontaining the recombinant plasmid pPGAPXSS HSODm is obtained. Thisplasmid is cut with SacI and the ca. 2 kb fragment containing GAPp,hSODm and GAPt (p-promoter, t-terminator) isolated by preparativeagarose gel electrophoresis and ligated to SalI cut and phosphatasedpC1/1 XSS. A colony containing the recominant pC1/1XSSGAPhSODm-PAplasmid with GAPP proximal to the ampicillin resistance gene wasobtained. The plasmid DNA was prepared and used to transform yeaststrain Saccharomyces cerevisiae P017 (Mata, leu2-04,cir^(°), selecting for colonies on agar plates lacking leucine.)

Example V Expression of hSODm in Yeast

[0080] Cultures are grown in 1 ml Leu⁻ medium to saturation and 1 μl ofthis culture was added to 1000 μl YEPD+0.2 mM MnCl₂ and grown tosaturation. Cells are harvested, lysed with glass beads, and theequivalent of 0.5 OD₆₅₀ units of cells loaded on a native SOD activitygel to determine activity.

Example VI Treatment of Patients with hSODm

[0081] Methods for treating patients with hSODm are similar to methodsfor treating patients with bovine SOD except that hSOD is expected to besomewhat more effective and may be capable of being used in reducedamounts or for shorter periods of time.

[0082] Inflammatory Joint Disease Patients

[0083] Patients having osteoarthritis or rheumatoid arthritis have beentreated by an intraarticular injection of 4 mg of bovine SOD and 8 mg ofsucrose in 2 ml of normal saline in an affected joint for six weeks, asdescribed in Flohe, supra. It is expected that 4 mg hSODm and 8 mgsucrose diluted in 2.0 ml normal saline will be effective whenadministered as a weekly intraarticular injection for four weeks.

[0084] Treatment of Patients Having Surgically Induced Ischemia

[0085] A solution containing 250 mg of hSOD and 500 mg sucrose in 500 mlphysiologic saline is expected to minimize reperfusion organ damage whenused to perfuse a donated kidney prior to irrigation of the kidneyduring kidney transplant surgery. Intravenous or intraarterialadministration as a bolus dose or continuous infusion of 50 mg to 500 mgtotal per patient during or following surgery is also expected tominimize reperfusion tissue damage.

[0086] The above results demonstrate the successful isolation andmanipulation of the human manganese superoxide dismutase codingsequence, its replication in microorganisms, its manipulation to provideexpression constructs for both prokaryotes and eukaryotes, particularlymicroorganism hosts, and the expression of functional human manganesesuperoxide dismutase in foreign hosts. Thus, a stable reproduciblesupply of human manganese superoxide dismutase is provided. In addition,contructs are manipulated to ensure that the superoxide dismutase isfree of the additional methionine initiation codon which is not found innatural human manganese superoxide dismutase. High yields of the enzymemay be obtained in cultures substantially free of materials which mighthave an adverse effect when the subject enzyme is employedtherapeutically.

[0087] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A polypeptide having substantially the same aminoacid sequence as human manganese superoxide dismutase, resulting as theexpression product of an expression construct in a unicellularmicroorganism.
 2. A polypeptide according to claim 1, wherein saidunicellular microorganism is a bacterium.
 3. A polypeptide according toclaim 2, wherein said bacterium is E. coli.
 4. A polypeptide accordingto claim 1, wherein said unicellular microorganism is a yeast.
 5. Apolypeptide according to claim 4, wherein said yeast is S. cerevisiae.6. A polypeptide according to claim 1, having other than lysine andhistidine as the N-terminal amino acid.
 7. A polypeptide according toclaim 6, wherein said N-terminal amino acid is methionine, serine,alanine, glycine, proline, threonine or valine.
 8. A polypeptideaccording to claim 7, wherein N-terminal amino acid is serine.
 9. Apolypeptide according to claim 7, wherein said unicellular microorganismis a bacterium.
 10. A DNA construct functional for expression in amicroorganism host comprising in downstream or order of transcription:(1) a transcriptional and translational initiation regulatory regionfunctional to said host; (2) an initiation codon; (3) a structural genecoding for a functional human manganese superoxide dismutase (hSODm)enzyme in reading frame with said initiation codon and having a stopcodon at the 3′-terminus; and (4) a transcription terminator.
 11. A DNAconstruct according to claim 10, wherein said initiation regulatoryregion and said initiation codon are separated by a minigene comprisingin the direction of transcription a second initiating codon, aShine-Dalgarno sequence included in a coding sequence and a stop codon,wherein said Shine-Dalgarno sequence is in functional relationship withsaid hSODm imitation codon.
 12. A DNA construct according to claim 10,joined to a replication system for extrachromosomal replication andmaintenance in said microorganism.
 13. A DNA construct according toclaim 10, wherein said microorganism is a bacterium.
 14. A DNA constructaccording to claim 11, wherein said bacterium is E. coli.
 15. A DNAconstruct according to claim 10, wherein said microorganism is a yeast.16. A DNA construct according to claim 15, wherein said yeast is S.cerevisiae.
 17. A method for preparing a first peptide havingsubstantially the same amino acid sequence and enzymatic activity ofhuman manganese superoxide dismutase (hSODm) and further characterizedby having an amino acid other than methionine at its N-terminus, saidmethod comprising: growing a unicellular microorganism transformant inan appropriate nutrient medium for a sufficient time for said peptide tobe expressed, where said transformant comprises a construct having inthe direction of transcription, a transcriptional and translationalregulatory region functional in said host, a structural gene encodingsubstantially the same amino acid sequence as hSODm but differing in atleast having a 5′-terminal methionine codon and the adjacent codoncoding for serine, alanine, glycine, proline, threonine or valine, andtranscriptional and translational termination region functional in saidhost, whereby said structural gene is expressed to produce a precursorpeptide which is processed by said host to remove the N-terminalmethionine; and isolating said first peptide.
 18. A method according toclaim 17, wherein said structural gene lacks the codons at the5′-terminus of the sequence for the mature hSODm for lysine andhistidine which are present in the wild-type structural gene.
 19. Amethod according to claim 17, wherein said transformant is E. coli. 20.A method according to claim 17, wherein said transformant is yeast. 21.A cDNA in substantially pure form coding for human manganese superoxidedismutase.
 22. A method of treating a patient having inflammatory jointdisease comprising intraarticularly injecting into a joint afflictedwith inflammatory joint disease a solution comprising human manganesesuperoxide dismutase in an amount effective to reduce inflammation in apharmaceutically acoceptable diluent for a period of time sufficient toreduce inflammation.
 23. The method of claim 22 wherein one mg to ten mgof human manganese superoxide dismutase is administered for two to eightweeks.
 24. The method according to claim 22 wherein said patient hasosteoarthritis or rheumatoid arthritis.
 25. The method according toclaim 22 wherein said solution comprises two mg to six mg humanmanganese superoxide dismutase in a suitable diluent.
 26. The methodaccording to claim 25 wherein said suitable diluent is physiologicsaline.
 27. The method according to claim 22 wherein said solution isinjected for four to six weeks.
 28. A method of treating a patient tominimize post-ischemic tissue damage comprising administering a solutioncomprising 10 mg to 1000 mg of human manganese superoxide dismutase in asuitable diluent to said patient during or following ischemia.
 29. Themethod according to claim 28 wherein said solution comprises 50 mg to500 mg human manganese superoxide dismutase in a suitable diluent. 30.The method according to claim 28 wherein said post-ischemic damageresults from disease and said solution is administered intravenously orintraarterially as a bolus dosage or a continuous infusion.
 31. Themethod according to claim 28 wherein said post-ischemic damage resultsfrom a surgical procedure and said solution is administered during saidsurgical procedure.
 32. The method according to claim 31 wherein saidpost-ischemic damage results from clamping an organ during surgery andsaid solution is administered directly into said organ prior toreirrigation of said organ.